Reader device for luminescent immunoassays

ABSTRACT

The present disclosure, among other things, describes a reader system comprising a casing, an optical system, en electromechanical motor system, and one or more digital processors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 61/510,779, filed Jul. 22, 2011, the entirety of which is herebyincorporated herein by reference.

BACKGROUND

Immunoassays may be used for the determination of clinical decisions. Assuch, accuracy, reliability, and repeatability of immunoassayinterpretation are largely important. With regard to the field of use,this analysis is important in a point-of-care or mobile setting. Inthese settings, tests may be carried out by unskilled technicians orpatients themselves, while still requiring the maintenance oftraceability and accuracy. In addition, communication of test andauditing data is important; insofar as it may be, for example, examinedby remote healthcare professionals, integrated with hospital LIMSsystems, or used to verify device operation.

SUMMARY OF CERTAIN EMBODIMENTS

Various embodiments of the present invention utilize photoluminescencebased methodologies to provide accurate, non-subjective interpretationof labelled mobilizable reagent binding in immunoassays, andquantization of analyte presence or concentration in fluid test samples.Bespoke algorithms and devices compensate for assay and opticalvariability. Embodiments of the invention combine portability,automation and communication technologies to cater for use by anunskilled technician in a point-of-care setting.

Embodiments of the present invention concern an immunoassay analysissystem for the recording and interpretation of photoluminescentimmunoassays, herein termed the reader system. Embodiments of theinvention quantify photoluminescence from one or more capture zones ofan immunoassay, and thereby determines a quantitative or qualitativemeasurement of analyte presence within a fluid sample.

In brief, a reader system according to embodiments of the presentinvention may comprise a casing, an optical system, en electromechanicalmotor system, and one or more digital processors. The casing may includeat least one port leading to a holster which is configured to receive acartridge comprising a vertically oriented immunoassay device foranalyzing one or more analytes in a fluid sample. The optical system mayinclude excitation optics comprising a light source and an excitationlens configured to transmit light from the light source and therebyexcite a region of the vertically oriented immunoassay device when acartridge is placed in the holster, and collection optics comprising aphotosensor and a collection lens configured to collect emitted lightfrom the vertically oriented immunoassay device when a cartridge isplaced in the holster. The electromechanical motor system may beconfigured to move the holster in a vertical direction with respect tothe optical system so that the optical system can interrogate differentregions of the vertically oriented immunoassay device when a cartridgeis placed in the holster. The one or more digital processors may beassociated electronics configured to receive data from and control theoptical system and to control the electromechanical motor system

A reader system, in some embodiments, includes non-volatile or volatiledigital memory for storing data generated by the optical system.

In some embodiments, the casing of a reader system further includes adisplay screen, a data entry device, such as a keypad or displayintegrated touch-screen, or a combined device that acts as a displayscreen and a data entry device.

In some embodiments, a light source is a light emitting diode (LED)surface mounted device. The light source may also include integratedlens for collimation of LED emitted light. In some embodiments, a readersystem includes multiple excitation sources, for example, LEDs withvarious central emission wavelengths, with matched optical filters.

In some embodiments, excitation optics includes an plate (e.g.,absorptive or reflective plate) with an optical aperture. The aperturemay be aligned within the optical excitation path; and defined to form aspecific, regular excitation area upon the immunoassay device. In animplementation, a excitation area is 0.3 mm-3 mm in width and 0.2 mm to2 mm in height.

In some embodiments, a collection lens collects emitted light from anentire excited region. The collection lens may integrate the emittedlight and direct it onto the central portion of a correspondingphotosensor for detection.

In some embodiments, excitation or collection optics each includes anoptical filter. For example, the optical filer can be a band-pass orshort-pass optical filer. The optical filer may operate by interferenceor by absorption. An optical filter in the excitation optics tunesoptical excitation wavelengths experienced by an immunoassay. An opticalfilter in the collection optics passes wavelengths associated with thephotoluminescent label emission of an immunoassay, while blockingwavelengths associated with optical excitation. Additionally oralternatively, an optical collection filter may be mechanicallyactuated.

In some embodiments, a cartridge comprising a vertically orientedimmunoassay device is located within a holster. The vertically orientedimmunoassay device may include one or multiple parallel, verticallyoriented immunoassay channels. The one or more immunoassay channels mayeach independently comprise one or more test lines for analyzing one ormore analytes. A reader system may include multiple light sources andmultiple photosensors, with one of each being dedicated to eachindividual immunoassay channel. The multiple light sources may each havedifferent central wavelength. Electronic frequency filtering may be usedto filer signals from the multiple photosensors. For example, electronicfiltering may be applied to the photosensor signal to register signalsassociated with the duty cycle of immunoassay excitation and thus theimmunoassay photoluminescence, while blocking low or high frequencysystem noise.

In certain embodiments, the pairs of light sources and photosensors areconfigured so that different immunoassay channels are interrogated atdifferent points in time. In certain embodiments, the multipleimmunoassay channels are spatially separated such that cross talkbetween different immunoassay channels and different photosensors issubstantially absent when the multiple immunoassay channels areinterrogated simultaneously.

In some embodiments, an aperture plate includes an aperture for eachlight source. There may be a dedicated collection lens associated witheach immunoassay channel and photosensor. The plate may be absorptive orreflective. At least one dimension (e.g., width, or height) of each ofone or more apertures is in a range of 0.1-2 mm, 0.7-0.8 mm, or 0.3-0.4mm.

In some embodiments, optical emission intensity of the light source iscontrolled and stabilised through a cartridge scan. In this case, theexcitation optics may include a dedicated excitation source monitoringphotosensor. The light source emission intensity can be monitored byanalysis of the monitoring photosensor electronic signal. Feedback ofthis monitoring signal to the excitation source may act to stabilize theemission of the excitation source across all scans. Feedbackstabilization may be carried out throughout a scan, for each duty cycleof each light source's emission. Alternatively, feedback stabilizationmay be independently carried out for each light source prior to thecommencement of each cartridge scan. In certain embodiments, a readersystem includes a proportional-integral-derivative control algorithm tooptimally stabilise light source emission at a desired intensity byanalysis of the monitoring photosensor signal.

In some embodiments, there is an angular offset between the opticalcollection plane of the photodiode/lens assembly, and the opticalexcitation plane. The specific angular offset and configuration can beselected in order to inhibit direct reflection of excitation light intothe detector assembly, while maintaining efficient excitation andcollection. For example, the optical excitation is normal to thecartridge surface, while detection is offset by 35 degrees.

In some embodiments, one or more digital processors collect datagenerated when the optical system scans the vertically orientedimmunoassay device. One or more digital processors can process the datato quantify an amount of one or more analytes in a fluid sample that wasapplied to the immunoassay device before the cartridge was placed in theholster.

For example, digital processors may use an algorithm to characterise thepresence or amount of an analyte within a fluid sample, according toassay specific calibration parameters. An algorithm provides either aquantitative, semi-quantitative or qualitative estimate of analyteconcentration within the fluid sample. In certain embodiment of thisinvention, multiple photoluminescent immunoassays assays are present inthe cartridge device, and the algorithm provides independentquantitative, semi-quantitative or qualitative estimations of analyteconcentration for all tested analytes within the sample.

In some embodiments, a reader system includes one or more qualitycontrols checks that are actualised in software on the one or moredigital processors. Quality controls checks may include: a qualitycontrol check of scan data, including a check of control linedevelopment; a check of channel clearance; and checks as to the size andposition of peaks. Additionally, the reader software verifies the timeof test as being within the expiry date of a particular assay.

In some embodiments, a reader system includes a barcode reading system.A barcode may be encoded on a cartridge. A barcode may be encoded withassay specific calibration data relating to an assay cartridge batch.Upon introduction of the cartridge to the reader system, the assayspecific calibration data may be read, interpreted and copied tointernal reader memory. For example, a barcode reading system may be onedimensional, or two dimensional. Exemplary information that can beencoded in a barcode reading system includes, but is not limited to,identification of cartridge type or lot data, lot manufacture and expirydates, analyte names, cartridge expected response, lot parameters, peakfinding parameters, calibration parameters, and any combination thereof.

In some embodiments, a read system includes at least one sensor forrecognizing cartridge insertion or removal. The sensor may be an opticalor mechanical sensor. For example, one or more optically emissivesources and corresponding optical sensors can be included. These sensorsmay be held within the cartridge holster, and their positioningcorresponds to locations which define the cartridge insertion or removalof the cartridge. In this case, the optical sensor may be a light sourceand photosensor couple. This may be located in close proximity to themouth of the holster. Upon insertion, the cartridge blocks propagationof light from the sensor light source to its corresponding photosensor.The sensor registers full removal of the cartridge by the resumption oflight propagation from the sensor light source to its correspondingphotosensor. A mechanical switch sensor may be located at the base ofthe holster. Upon full insertion of the cartridge into the holster, thisswitch is actuated by the cartridge, enabling the detection of cartridgeinsertion. In some embodiments, there exists a physically separatequality control component with substantially the same externaldimensions as a cartridge. This component may include photoluminescentmaterials that exhibit characterised levels of photoluminescence uponexcitation by the light source. For example, photoluminescent materialscan be or comprise plastics impregnated with photoluminescent dyes,nanocrystals, quantum dots or any combination thereof. Generally, thephotoluminescent areas of a quality control component are localised atthe optical plane of the reader, at a similar position to that ofimmunoassay surface in a given cartridge. Further, reader scans of thisquality control component may be carried out in a similar method to thatof the immunoassay cartridge. Photoluminescent areas may be defined onthe quality control component using masked materials, coated materials,multilayer etched materials or any combination thereof. Photoluminescentareas may be patterned such that optical misalignments within the readersystem lead to predictable changes in the pattern or intensity ofemitted light.

In some embodiments, the one or more digital processors of a read systemcollect data generated when the optical system scans the quality controlcomponent and use the data to validate the reader system for furtheruse. The one or more digital processors may collect data generated whenthe optical system scans the quality control component and use the datato modify internal calibration factors of the reader system. The one ormore digital processors may collect data generated when the opticalsystem scans the quality control component and use the data to calculatea degree of optical misalignment, such as the direction and degree oflateral misalignment, degree of focus or defocus, or optical systemtilt, between the optical system and the holster. In this case, the oneor more digital processors may control the electromechanical motorsystem to actuate and thereby bring the optical system and the holsterinto optical alignment.

In some embodiments, a reader system includes travel sensors that areused to detect and report a relative position of the holster. The travelsensors may be optical or mechanical.

In some embodiments, a reader system includes a printer for the printoutof hardcopies of scan results and data following the reading of animmunoassay cartridge, or data from stored memory. This printer may beincorporated within the casing of the reader device, or provided as a aseparate component. In the case of the printer being a separatecomponent, the printer may be interfaced with the reader using a USB,Ethernet or serial port connection. Power may be provided to the printerdevice directly from the reader system or via a separate power supplycomponent.

In some embodiments, a reader system includes a processing algorithm forthe verification of immunoassay batch responses. This algorithm analysesthe response of one or more immunoassay cartridges of the specifiedbatch, run with control liquids of specified concentrations. Thealgorithm compares expected responses with those found from thesecartridges, and verifies the immunoassay batch as operating to a givenspecification. Further, this algorithm also may conduct the optimisationand correction of immunoassay batch specific calibration parameters, asstored within reader memory, to compensate for time-related changes inimmunoassay photoluminescence response. In this case, following theanalysis of one or more immunoassay cartridges of the specified batch,run with control liquids of specified concentrations, internalcalibration parameters are then updated to provide a best-fit result tocontrol responses.

In some embodiments, a reader system includes components and protocolsfor external wireless access, such as by Wi-Fi, ANT or Bluetooth. In animplementation, this connectivity enables remote reader operationdiagnostics, firmware or software updates and data transfer.

In some embodiments, a reader system includes components and protocolsfor wired connectivity, such as by RS-232 serial, universal serial bus(USB) or Ethernet cable. In an implementation, this connectivity enablesremote reader operation diagnostics, firmware or software updates anddata transfer.

In some embodiments, a reader system includes alignment features withina cartridge holster. These features hold the cartridge in position andensure that the immunoassay surface is localised at the optical plane.In certain embodiments, spring loaded dowels are located in positionscorresponding to recesses in the assay cartridge when the cartridge iscorrectly localised within the holster. In certain embodiment, physicalalignment features prevent the mis-insertion of the cartridge, byblocking full insertion of the cartridge at an incorrect rotation.

In some embodiments, a reader system includes an internal battery whichsupplies the reader with electrical power when not connected to a mainspower supply.

In some embodiments, a reader system includes electronic memory and adigital file management system for the storage of data, operationparameters, and software and user interface details. Files stored withinthis electronic memory may include: Scan files, calibration files,quality control run files, user lists, settings and change logs, scanlogs, calibration run logs, or user logs. In order to review apotentially large number of scan files, search functionality may beimplemented. This search functionality generally consists of userinterface options enabling the user to filter scans results by date,operator, patient ID or test.

The present disclosure include, among other thing, methods of usingreader systems described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reader system top level diagram.

FIGS. 2(a) and 2(b) show reader system casing diagrams: (a) side A and(b) side B.

FIGS. 3(a) and 3(b) show reader optics diagrams.

FIG. 4 shows a reader optics diagram for a multiple wavelength system.

FIG. 5 shows a reader use procedure block diagram for a test scan.

FIG. 6 shows a reader use procedure block diagram for a quality controlscan.

FIG. 7 shows a reader use procedure block diagram for liquid controlsscans.

FIG. 8 shows a scan processing algorithm block diagram.

FIG. 9 shows a test scan calibration algorithm block diagram.

FIG. 10 shows a quality control algorithm block diagram.

FIG. 11 shows a liquid controls calibration adjustment algorithm blockdiagram for a qualitative tests.

FIG. 12 shows a liquid controls calibration adjustment algorithm blockdiagram for a quantitative test.

FIG. 13 shows an example printout of test data following a test scan.

FIG. 14 shows a schematic of sample light source emission power feedbackalgorithm.

FIGS. 15(a) and 15(b) show exemplary optical-path schematics of a readersystem: (a) side view and (b) top view.

FIG. 16 shows an example optical scan taken by an reader system.

DEFINITIONS

Assay—As used herein, the term “assay,” refers to an in vitro analysiscarried out to determine the presence or absence of one or more targetanalytes in a fluid sample. In certain embodiments the assay may bequantitative and determine the amount of the one or more target analytesin the fluid sample. In general, an assay includes at least one pair ofreagent components where at least one of the reagent components has ahigh binding affinity for the other. In certain embodiments, the assayis an immunoassay (e.g., a sandwich, competitive or inhibitionimmunoassay). Generally, an immunoassay includes an antibody componentwhich binds with high affinity to another antibody component or to anantigen component. In certain embodiments, the assay is a molecularassay and includes a pair of nucleic acid components which hybridize toform a complex.

Target analyte—As used herein, the term “target analyte” or “analyte”refers to the substance or substances that an assay is designed todetect. Examples of analytes include, but are not restricted to proteins(e.g., antibodies, hormones, enzymes, glycoproteins, peptides, etc.),nucleic acids (e.g., DNA, RNA, etc.), lipids, small molecules (e.g.,drugs of abuse, steroids, environmental contaminants, etc.) andinfectious disease agents of bacterial or viral origin (e.g., E. coli,Streptococcus, Chlamydia, Influenza, Hepatitis, HIV, Rubella, etc.).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

A reader system for the recording and interpretation of photoluminescentimmunoassays is described herein in connection with embodiments of thepresent invention. Various embodiments of the invention quantifyphotoluminescence from one or more capture zones of said immunoassay,and determines a quantitative or qualitative measurement of analytepresence within a fluid sample.

In brief, a reader system may include a casing, incorporating a displayscreen, or a data entry device, such as a keypad or display integratedtouch-screen, or a combined device that acts as a display screen and adata entry device. This casing may also incorporate a fluidicimmunoassay device access port, and contains a holster receptacle forreceiving an external immunoassay fluidic device. Said externalimmunoassay fluidic device comprises one or more immunoassays orientedin a substantially vertical configuration, configured for the analysisof a fluid sample; and is herein referred to as the cartridge. Further,the reader comprises an optical system within the casing, consisting ofexcitation and collection optics within a single optical block; and anelectromechanical motor system, such as a stepper motor, whereby theholster is moved in a vertical direction with respect to the opticalblock. Also, the reader system incorporates a digital processor andelectronics for the actuation and control of readings, and non-volatiledigital memory for the storing of data. It is to be understood that thereader system may incorporate multiple processor components, with adivision of processing occurring between separate components. Forexample a single processor may handle sensing and time critical tasks,while an additional processor may control screen display, user interfaceoperations, communications, and additional processing. Additionally, thereader may incorporate communication ports or wireless connectivity,internal batteries and an internal or external printer unit (such as athermal printer) for the printout of hardcopies of scan resultsfollowing the reading of an assay, or from stored memory.

With regard to the particulars of the various reader assemblies andcomponents, these are further detailed, below.

In some embodiments, the cartridge holster of a reader system includesalignment features to ensure the correct insertion and placement of thecartridge within the reader. In certain embodiments, spring loadeddowels are located in positions corresponding to recesses in the assaycartridge when the cartridge is correctly localised within the holster.Upon correct insertion of the cartridge, the dowels register withrecesses in the cartridge. This locks the cartridge in position, andensures that the immunoassay surface is localised at the optical planeuntil a force is applied to remove said cartridge. In certainembodiments, physical alignment features prevent the mis-insertion ofthe cartridge, by blocking insertion of the cartridge at an incorrectrotational alignment. In certain embodiments, the cartridge holsterincorporates fluidic flow channels to ensure any liquid spillages fromthe cartridge or into the reader port flow along a defined path to aspill receiver area. This area may be upon, or otherwise connected to anadditional removable cover on the underside of the reader casing,enabling access to and cleaning of this spill area without disassemblyof the reader device.

In some embodiments, a reader system includes optically emissive sourcesand corresponding optical sensors for the recognition of cartridgeinsertion. These sources and sensors can generally be placed within thecartridge holster opposite the optics block. The positioning of theseoptical components corresponds to absorptive or reflective features uponthe cartridge, generally defined on the distal surface of the cartridgeto that of the assay channels. Upon insertion of the cartridge into thecartridge holster, the response registered by the optical sensors variesas light emitted by the optically emissive sources interact with theabsorptive or reflective features upon the cartridge. Analysis of thesensor response during the insertion of the cartridge enablesrecognition of the cartridge movement direction, and verification offull cartridge insertion. Additional absorptive or reflective featuresmay be incorporated upon the cartridge may, encoding informationrelating to the identification of cartridge type or immunoassaycartridge batch data. In this case, the reader may also incorporateadditional optically emissive source and corresponding optical sensorcomponents for the registration of these features. In certainembodiments, the reader system may incorporate optical or mechanicalsensors, which register full cartridge insertion or removal. In thiscase, the reader may automatically initiate travel of the holsterwithout further user intervention upon full cartridge insertion orremoval. For example, the reader system may initiate a scan or move ofthe holster to a rest position, upon insertion or removal of thecartridge, respectively.

In some embodiments, a reader system includes a one dimensional or twodimensional barcode reader, as known in the art, within the readercasing. Generally, these register and read barcode structures disposedupon the assay cartridge. In an embodiment of this invention, thebarcode encodes information for the identification of cartridge type orlot data. In certain embodiments, the barcode is a two dimensionalbarcode and encodes information corresponding to any of the following:identification of cartridge type or lot data, lot manufacture and expirydates, analyte names, cartridge expected response, lot parameters, peakfinding parameters, and calibration parameters for the immunoassaycartridge batch.

In some embodiments, a reader system includes a radio frequencyidentification (RFID) reader. This registers and reads RFID chipspresent in or on the assay cartridge. In an embodiment of thisinvention, the RFID chip encodes information relating to any of:identification of cartridge type or lot data, lot manufacture and expirydates, analyte names, cartridge expected response, lot parameters, peakfinding parameters, and calibration parameters for the cartridge lot.

In some embodiments, the motor component integrates an encoder systemwhich detects and reports the relative motor actuation position.Alternatively, holster relative position may be determined bycalculation from motor speed and time of travel. In each case, holsterposition may be determined with reference to this relative measurementand signals received from particular optical or mechanical travelsensors located relative to specific positions in the holster travel. Inthe case of optical travel sensors, the holster component incorporatesbeam blocking features, which break an optical beam sensed by theoptical travel sensors, indicating holster position at these locations.Alternatively, the holster component may incorporate reflectivefeatures, which direct an optical beam to the optical travel sensors,indicating holster position at these locations.

An example reader system optical block and optical paths isschematically represented in FIGS. 15 (a) and 15 (b). In an embodimentof the present invention, the excitation optics within the optical blockinclude: one or more light sources [302] and an excitation lens [305].In particular embodiments of the present invention, an excitation lightsource may comprise any of, for example: an inorganic light emittingdiode (LED), or an organic LED, or a laser. Generally, the light sourceshave emission wavelengths compatible with the excitation spectra ofphotoluminescent labels associated with the mobilizable or controlreagents of the assay. In an embodiment of the present invention,optical excitation is derived from six surface mounted device LEDs, eachwith an integrated lens which serves to partially collimate emittedlight.

Generally, one or more excitation lenses [305] are located within thelight paths of excitation, and direct light source emitted opticalenergy to the surface of the immunoassay device. An excitation lens mayalso act to collimate or spread excitation light. An excitation lens maybe formed of one of a variety of optically transparent materials;including glass, fused silica or organic polymers (for example:polymethylmethacrylate, polycarbonate, polytetrafluoroethylene,polystyrene, or cyclic olefin co-polymer). Generally, an excitation lensis of a light converging form, with the design of the lens being one of,for example: convex, bi convex, spherical, plano-convex, positivemeniscus, or aspheric. Lens parameters include focal length, numericalaperture, material and optical coatings. These are selected to optimisethe optical design and have transparency corresponding to thewavelengths of excitation.

In particular embodiments of the reader system, excitation opticsincorporate an absorptive or reflective plate with one or more opticalapertures [303]. The aperture plate may be coated with a stableabsorptive material to ensure scattering and reflections are limited.Defined areas of stable diffusely reflective material may be furthercoated onto the aperture plate, causing a portion of light sourceemitted light to be back-reflected to light-source monitoringphotosensors. Optical apertures restrict excitation light rays to thosepassing through the aperture. Each aperture is aligned to an opticalexcitation path of a single corresponding light source, and is shapedand sized to block specific light rays. An aperture forms a specific,regular excitation area upon the immunoassay device by blocking lightcorresponding to optical rays which would illuminate sections outsidethis area. The placement and size of this optical excitation area may betuned by modifying the corresponding aperture's position and dimensions.Apertures may be designed to ensure that all excitation areas areregular and of similar size. In particular, the aperture plate may becurved, ensuring optical light paths are of similar lengths followingpassing through the aperture plate. In particular embodiments, aperturesact to collimate the excitation light by selecting light raysoriginating from the central, more homogenous angles of light sourceemission. This may be important for subsequent optical filtering usingan interference type filter, as the pass and stop bands of these filtersare dependent on the angle of incidence of a light beam. In certainembodiments, apertures are 0.2 mm-2 mm in width and 0.1 mm to 1 mm inheight, and each excitation area is 0.3 mm-3 mm in width and 0.2 mm to 2mm in height.

In particular embodiments of the present invention, excitation opticsalso comprise one or more optical filters [304]. An optical excitationfilter shapes the spectral profile of excitation light experienced bythe immunoassay device. This filter may act to ensure spectralseparation between excitation light and luminophore emitted light. Anoptical excitation filter may, for example, be of band-pass orshort-pass variety, and may operate by interference or absorptivemechanisms. Generally, an excitation filter is selected such that thefilter pass-band corresponds to some portion of the excitation spectrumof the photoluminescent labels associated with the mobilizable orcontrol reagents of the assay, and that the filter stop-band correspondsto some portion of the emission spectrum of these photoluminescentlabels. The Stokes' shift between a photoluminescent label's excitationand emission spectra defines the maximum filter transition band. In anembodiment, a short pass interference optical filter is selected.

Generally, collection optics include: one or more collection lenses[307] for the collection of light emitted from the immunoassay surface;and one or more photosensors [306] for the detection and transduction ofthis luminescence to an electrical signal.

Generally, each photosensor is a device which transduces optical energydirected at the surface of this sensor into an electrically registeredsignal. Photosensors are selected to be responsive to optical emissionwavelengths of the photoluminescent assay labels. These photosensors maybe selected from, for example: photodiodes, phototransistors,photo-resistors, charge coupled devices, or photon multiplier tubes.

In an embodiment of the present invention, a collection lens is locatedwithin the collection light path and collects light emitted from anexcitation area, that area of the immunoassay surface illuminated by theexcitation optical assembly, and directs this light towards acorresponding photosensor. In a specific embodiment of the presentinvention, the collection lens directs light emitted from the fullexcitation area to the central portion of a corresponding photosensor.In this case, selection of the size of the excitation area and theintegration of the optical signal over the full excitation area iscarried out to afford resilience to local inhomogeneity in the assaymaterials. Also, as light is directed towards the centre of the sensor,the system may withstand some misalignment of the optics before light istransferred to an area outside the active area of the photosensor. Acollection lens may be formed of one of a variety of transparentmaterials; including glass, fused silica or organic polymers (e.g.,polymethylmethacrylate, polycarbonate, polytetrafluoroethylene,polystyrene or cyclic olefin co-polymer). Generally a collection lens isof a light converging form, with the design of the lens being one of,for example: convex, bi-convex, spherical, plano-convex, positivemeniscus or aspheric. Lens parameters include focal length, numericalaperture, material and coatings. These are selected to optimise theoptical design and conform to the wavelengths of collection.

In particular embodiments of the present invention, collection opticsalso comprise one or more optical filters [308]. Such a filter islocated within the optical collection path. The optical collectionfilter shapes the spectral profile of collected light prior to detectionby the photosensor. This filter acts to ensure that residual excitationlight is not transmitted to the photosensor. An optical collectionfilter may, for example, be of band-pass or long-pass variety, and mayoperate by interference or absorptive mechanisms. The filterspecification is selected to have a stop-band including the spectralbandwidth of the excitation light source, following optical filtrationby any excitation filter, and a pass-band including some portion of theemission wavelengths of the photoluminescent labels. In an embodiment, along pass absorptive optical collection filter is selected. In theparticular case of an interference-type collection filter, an additionalcollection lens may be present in the reader device. In this case, thefirst collection lens may be located within the collection light pathand collects light emitted from an excitation area, that area of theimmunoassay surface illuminated by the excitation optical assembly, andacts to collimate or partially collimate this light. Theinterference-type collection filter can be placed between this firstcollection lens, and the second collection lens. The second collectionlens can direct the filtered light towards a corresponding photosensor.In a particular embodiment of the present invention, there may be anangular offset between the plane of optical collection paths of thecollection optics, and the plane of optical excitation paths of theexcitation optics. The angular position of these planes and theirspecific offset can be selected in order to inhibit direct reflection ofexcitation light into the detector assembly. In an embodiment, as shownin FIG. 15(a), the optical excitation plane is normal to the cartridgesurface, while detection is offset by 35 degrees.

In particular embodiments of the present invention, the reader system iscapable of selecting one of multiple optical wavelength bands foroptical excitation of the sample, corresponding to the excitationwavelengths of one of multiple photoluminescent labels associated withthe mobilizable or control reagents of immunoassays. Likewise, thereader system is capable of selecting one of a variety of variousoptical wavelength bands for detection of the optical signalscorresponding to the emission wavelengths of one of multiplephotoluminescent labels associated with the mobilizable or controlreagents of immunoassays. In this regard, the reader may incorporatemultiple excitation sources; consisting of multiple banks of LEDs orother excitation light sources, each bank emitting optical radiation ata particular central wavelength. The specific bank of excitation sourcesused for optical excitation of the sample during a scan is selected withregard to the excitation maxima of the photoluminescent labels. Eachbank of excitation light sources may have an associated opticalexcitation filter and aperture plate. The banks of excitation lightsources may be placed at 90 degrees to one another, at similar distancesfrom the sample plane. Light emitted from one bank of excitation lightsources may be normal to the sample plane.

In an embodiment of the reader system, as shown in FIG. 4, two banks ofLEDs [401], [402] are used with directions of emission normal to andparallel to the sample surface. A half-reflective mirror [403] is placedin the excitation light paths at 45 degrees from each of the LED bank'snominal light paths, such that some portion of light emitted from eachbank is directed to produce excitation areas upon the immunoassaysurface at similar positions and dimensions. The reader's nominalexcitation wavelength is then selected by activating only the LED bankof this wavelength.

In an alternative embodiment of the reader system, two banks of LEDs areused, with directions of emission normal to and parallel to the samplesurface. A mechanically actuated mirror is placed such that it may bemoved into an “engaged” position within the excitation light paths, at45 degrees from each of the LED bank's nominal light paths. In thiscase, light originating from the LED bank with emission normal to thesample surface has its emission blocked, while that with emissionparallel to the assay surface has its light reflected to form excitationareas on the assay surface. Thus, actuation to the “engaged” positionensures excitation of the sample using a first wavelength. In a“non-engaged” position, the mirror is in neither of the optical lightpaths. In this case, light originating from the LED bank with emissionnormal to the sample surface forms excitation areas on the assaysurface, while light originating from the LED bank with emissionparallel to the assay surface does not reach the assay surface. Thus,actuation to the “non-engaged” position ensures excitation of the sampleusing the second wavelength.

In particular embodiments of the present invention, the reader mayselect between specific wavelength ranges of sensitivity in detection.In this regard, a mechanically actuated selection of optical filters ispresent within the collection assembly, such as a motor turned filterwheel. Selection of a particular filter ensures that this filter lieswithin the collection optical path for a particular scan. In thisregard, collected light with energies corresponding to the pass-range ofthis filter is transmitted to the optical detector, determining theoptical wavelength response of the reader. The particular filterselected in an assay scan is selected to ensure transmission of someportion of the light emitted by the photoluminescent labels associatedwith the mobilizable or control reagents of immunoassays, and exclusionof stray excitation light. Alternatively, multiple light collectionassemblies may be present within the reader system with various spectralsensitivities. For example, the reader system may incorporate a secondset of lens, filer and detector elements in a mirrored layout to thepreviously specified assemblies. This may be located at an angle abovethe plane of cartridge excitation. In this case the collection filtersmay be each long-pass or bandpass in character, and may be tuned to passsubstantially different wavelengths bands of light. By simultaneous, ortemporally separated monitoring of photodetector signals or eachdetector assembly emission from multiple spectrally separatedphotoluminescent labels may be distinguished within a single scan. Inthis regard, the reader instrument is able to address and separatelyregister multiple different sets of overlapping emissive features withina single channel.

In an embodiment of the present invention, the reader systemincorporates one or more digital processors and electronics for theactuation and control of readings. Generally, these control theoperation of motors, optical electronic components, display components,and scan processing. Digital processors also interpret data entry andcommunications protocols. Additionally, the digital processors controlany internal digital memory; enabling the writing, reading, search andtransfer of data. The digital processors carry out scan processing andinterpretation algorithms, and controls the various electroniccomponents of the reader devices.

In some embodiments, a reader system includes non-volatile or volatiledigital memory for the storing of data. Generally, such data may includecollected scan data, and corresponding patient details and assayresults; user details and passwords; events and error logs; calibrationparameters; reader settings; user interface screens; interface andcommunications parameters; and reader operation programs. This memorymay consist of one of, or multiple instances of, for example, internalflash memory, magnetic hard-drives, and SD-card components.

In some embodiments, a reader system includes one or more communicationsports. Components and protocols are incorporated for wired connectivity,such as universal serial bus (USB), Ethernet (IEEE 802.3), and serialrecommended standard 232 (RS-232). These facilitate communication todevices external to the reader, such as personal computers or mobiledevices. These may also enable control and powering of external devices,such as barcode readers or printers. These may also facilitateconnections to hospital or laboratory information management systems. Inan embodiment, this connectivity enables remote diagnostics, firmware orsoftware updates and data transfer, and control of an external barcodereader device.

In some embodiments, a reader system includes components and protocolsfor external wireless access, such as by Wi-Fi (IEEE 802.11), ANT orBluetooth. These facilitate communication to, or control of, devicesexternal to the reader. These may also facilitate connections tohospital or laboratory information management systems. In an embodiment,this connectivity enables remote diagnostics, firmware or softwareupdates and data transfer.

In some embodiments, a reader system includes a printer for the printoutof hardcopies of scan results and associated audit data following thereading of an assay, or from stored memory. Additional printable datamay include: user lists, reader settings, events or error logs,installed calibrations, quality control results, etc. This printer maybe of a type including: thermal, ink-jet, laser, or dot-matrix. In anembodiment, this reader is within the reader casing and is of thermaltype.

In some embodiments, a reader system is portable, being intended forbench- or table-top point-of-care use. In an embodiment of the presentinvention, the reader includes an internal rechargeable battery, whichmay power the reader in situations where the system is not connected tomains power supplies. This battery is rechargeable, and charges whilethe reader is connected to a mains power supply. Electronics and thedigital processor may monitor battery charge, reporting this to theuser, and regulating such details as: charge speed, battery temperature,and minimum charge levels before the unit is automatically shut down. Incertain embodiments of the reader system, batteries may be held in aremovable battery pack, or be insertable into a dedicated batterycompartment by the user. In certain embodiments, a reader systemincorporates a speaker for transmission of auditory alarms, or auditoryfeedback or user actions. In certain embodiments, the reader comprisesan internal clock. This clock is generally powered by a separate, longlife battery component.

In an embodiment of the present invention, a Secure Digital (SD) cardcomponent holds assay specific calibration data relating to an assaycartridge batch. The SD card may be introduced into the reader system,and the assay specific calibration data copied to internal readermemory. In an embodiment of the present invention, the SD card is asecure write-once, read many times form. This card may be encoded withidentification data corresponding to unique characteristics of theparticular card, enabling security of written data and recognition ofthe correct card type prior to transfer of information.

In an embodiment of the current invention, an SD card may hold firmwareor software updates for the reader device. Alternatively, a standard SDcard may be inserted into the SD card slot, and the user may transfersaved data (such as scans, results, settings, calibrations or qualitycontrol data) from the internal device to the SD card for back up orsubsequent transport.

In an embodiment of the present invention, the excitation sourceactivation and emission timings and photosensor read timings are tunedin accordance with the positions and numbers of assay channels withinthe immunoassay cartridge. These parameters may be stored in the batchcalibration file, and the excitation and read logic of the reader systemis modified with regard to the cartridge structure. For example, in areader system with six channels, the system is presented with a threechannel immunoassay cartridge. The reader system is informed of thepositions of the present assay channels, and acts to only excite andread from these channels, modifying the relevant timings accordingly.

In an embodiment of the present invention, the excitation sourceemission timings and photosensor read timings are tuned such that onlyone test is being excited at a specific time, ensuring that opticalcrosstalk between channels is minimised. Alternatively, multiplechannels may be illuminated and read simultaneously. However, thesechannels may be spatially separated in order to ensuring that opticalcrosstalk between channels is minimised.

In an embodiment of the present invention, electronic frequencyfiltering is applied to the photosensor signal. The pass-band of thiselectronic filter is tuned to the frequency of the excitation sourceduty cycle, and serves to amplify the detected photoluminescence whileattenuating noise signals. Such noise may be associated with constant(low frequency) light leakage into the system, or high frequencyelectronic noise. Such a filter may be comprised of multiple high-passand low-pass electronic filters placed in series.

In an embodiment of the present invention, a reader system records adark count, corresponding to detected signal from each channel withoutactivation of the corresponding light source for an equivalent time tothe defined excitation source emission time and at a time close to eachexcitation duty cycle. In this case, the photosensor signals may becorrected by subtraction of this dark count from that acquired duringactivation of the corresponding light source. This enables compensationfor light leakage into the device, interference from light generationwithin the device, or thermal or electronic noise.

In an embodiment of this invention, time resolved detection ofphotoluminescence may be employed in the reader system. In this regard,an excitation source may be activated briefly, and correspondingphotoluminescence recording initiated some time (generally at least tensto hundreds of nanoseconds) after the light source has been deactivated.In this manner, photoluminescence from long emissive lifetime (forexample, lanthanide labels with emissive lifetimes of multiplemicroseconds) photoluminescent labels may be discriminated from shorterlifetime background fluorescence (generally termed in nanoseconds).

In an embodiment of this invention, the light source optical emissionintensity is controlled and stabilised through the cartridge scan. Inthis case, the excitation optics incorporates a dedicated excitationsource monitoring photosensor. The light source emission intensity isthereby monitored by analysis of the monitoring photosensor electronicsignal Feedback of this monitoring signal to the excitation source mayensure that the that the emission of the excitation source remainsconstant across all scans. Feedback stabilization may be carried outthroughout a scan, across each duty cycle of each light source'semission. Alternatively, feedback stabilization may be independentlycarried out for each light source prior to the commencement of eachcartridge scan. In an embodiment of this invention, the readerincorporates a proportional-integral-derivative control algorithm tooptimally stabilise light source emission at a desired intensity byanalysis of the monitoring photosensor signal.

In some embodiments, a reader system uses an algorithm for the detectionof optical emission peaks from each optical scan. Algorithm parametersmay include such details as expected numbers of peaks, expected peakscan positions, expected widths of peaks, expected ranges of peakheights. Peak detection algorithms may include background subtraction;compensating for background fluorescence derived from the assaymaterials, stray background light, unbound labelled assay materials orother sources. This may be realised by subtracting the minima of a scan,or estimation and subsequent subtraction of background fluorescence atthe point of the peak maximum. In an embodiment, such an estimation iscarried out by registering fluorescence levels at particular scanpositions at a defined distance to either side of the peak position,then determining a linear fit to the background versus scan position,and finally estimating the level of background fluorescence at a scanposition at a position corresponding to the peak maximum.

In particular embodiments of the present invention, sets of qualitycontrols are actualised in software to ensure that the assay progressedin a defined manner. These may include: quality control checks of scandata, including a check of control line development, a check of channelclearance, and checks as to the size and position of peaks.Additionally, controls may verify the time of test as being within theexpiry data of a particular assay. In particular, the level of detectedluminescence is characterised at a particular scan position, defined incalibration parameters for the assay in question, at which no capture orcontrol zones are present, and which generally corresponds to backgroundfluorescence. If the magnitude of this photoluminescence is found to beabove a certain level defined in calibration parameters for the assay inquestion, the unbound luminescent materials is not taken to haveachieved full clearance, and the particular assay is termed a “Missrun”.Control zone peaks are further analysed: if these are not found, or areof insufficient magnitude, the assay is likewise is considered to havenot fully developed, and is likewise termed a “Missrun”.

In an embodiment of the present invention, the reader includes acalibration algorithm for the qualification or quantification of analytepresence within an immunoassay fluid sample. These algorithms take asinput the following: calibration parameters specific to the assay batchand peak heights as determined by a peak detection algorithm for each ofthe capture and control zones. For each analyte, the algorithm processesthe corresponding capture zone peak height, according to the calibrationparameters. Alternative algorithms may alternatively normalize thecapture zone peak height by the control zone peak height, compensatingfor flow related or assay component related variability. Generally forqualitative tests, the algorithm compares the peak height versus athreshold value, and reports a positive or negative result.Alternatively for quantitative tests, the algorithm characterises theconcentration of an analyte within the test sample, according to assayspecific calibration parameters. In this case, the algorithm may reportthat the concentration is greater or less than particular limits ofquantization, respectively. Finally, for semi-quantitative tests, thealgorithm characterises the concentration of an analyte within the testsample to be within specific ranges, according to assay specificcalibration parameters. It should be understood that a multiplex assaypanel may consist of a selection of qualitative, quantitative andsemi-quantitative assays, all within a single cartridge, being read andinterpreted simultaneously.

In the case where the assay batch includes parallel tests for aparticular analyte with similar sensitivities, an alternativecalibration algorithm may take as input the following: calibrationparameters specific to the assay batch and peak heights as determined bya peak detection algorithm for each of the capture and control zones foreach of the parallel tests. For each analyte, the algorithm processesthe corresponding capture zone peak heights, according to thecalibration parameters. Estimation error in the quantitative orquantitative estimate of analyte presence may be minimised by averagingmultiple results, or by discarding results with outlying values orcorresponding peak heights.

In the case where the assay batch includes parallel tests for aparticular analyte with varying sensitivities and corresponding linearranges, an alternative calibration algorithm may take as input thefollowing: calibration parameters specific to the assay batch and peakheights as determined by a peak detection algorithm for each of thecapture and control zones for each of the parallel tests. For eachanalyte, the algorithm processes the corresponding capture zone peakheights, according to the calibration parameters. The algorithm thenselects a result predicted by one test in which the quantitativemeasurement is within the linear range of the test. Additionally, wherethe measurement is within the linear range of multiple tests, thealgorithm may report the result as the weighted average of the analyteconcentrations estimated from each such test.

Finally, in the case where the assay batch includes various testsrelated to single or multiple clinical decisions, a secondary algorithmmay take as input the quantitative or qualitative estimates from eachindividual test as provided by the calibration algorithm. This secondaryalgorithm processes the various calibration results and reports a singlediagnostic result or multiple diagnostic result.

In an embodiment of the present invention, a physically separate qualitycontrol component, of external dimensions similar to that of the assaycartridge is incorporated. This component incorporates materialsexhibiting specific, characterised efficiencies of photoluminescenceupon optical excitation at a wavelength corresponding to the readerexcitation source. In an alternative embodiment of the currentinvention, the quality control component may be disposed on each assaycartridge, at a position separate from the assays. In another embodimentof the current invention, the quality control component may beintegrated within the reader system itself. In particular embodiments ofthe current invention, the quality control component may be integratedwithin the reader's cartridge holster, being automatically actuated toand from the optical plane upon removal and insertion of the testcartridge, respectively.

In embodiments of the present invention, the quality control component'sphotoluminescent areas are defined using masked photoluminescentmaterials, coated non-fluorescent materials or multilayer etchedmaterials. Photoluminescent materials may consist of plasticsimpregnated with fluorescent dyes, nanocrystals or quantum dots.

In embodiments of the present invention, the quality control component'sphotoluminescent areas may be localised at the optical plane within thereader. Photoluminescent areas are patterned in a defined manner, suchthat optical misalignments will lead to predictable changes in scanresponses.

In a first embodiment of the present invention, a processing algorithmfor the analysis of quality control component scans is incorporated.This algorithm will compare the expected response from fluorescent areaswith those of received responses, and validate the reader for theanalysis of assays. In a second embodiment of the present invention, thealgorithm will compare the expected response from fluorescent areas withthose of received responses, and compensate for changes in systemresponse by the modification of internal calibration factors. In a thirdembodiment of the present invention, the algorithm compares the readerresponse with the expected response from the patterned fluorescentcomponent. This algorithm then calculates the type and degree of opticalmisalignment, such as: the direction and degree of lateral misalignment,degree of focus or defocus, or optical system tilt. In this case, thereader may incorporate motor driven alignment of the optical stage.Reader algorithms analyse the quality control component scan, andautomatically adjust the position of the optical stage for optimalsystem alignment.

In an embodiment of this invention, the reader incorporates a processingalgorithm for the verification of immunoassay batch response. Thisalgorithm analyses the response of one or more immunoassay cartridges ofthe specified batch, run with control liquids of specifiedconcentrations. The algorithm compares expected responses with thosefound from these cartridges, and verifies the immunoassay batch asoperating to a given specification. Further, this algorithm also mayconduct the optimisation and correction of immunoassay batch specificcalibration parameters, as stored within reader memory, to compensatefor time-related changes in immunoassay photoluminescence response. Inthis case, following the analysis of one or more immunoassay cartridgesof the specified batch, run with control liquids of specifiedconcentrations, internal calibration parameters are then updated toprovide a best-fit result to control responses.

Embodiments of the present invention include procedures and methods forupdating reader software and firmware. In certain embodiments, an updatemay be initiated by the user selecting particular menu options of thereader's user interface. The reader system may receive datacorresponding to the compiled firmware or software code from a varietyof sources, including but not limited to: an SD card inserted into theSD card port, a USB flash drive inserted into a USB port, an externalconnected personal computer, or a wireless connection. In certainembodiments of the reader system, firmware or software updates have“roll-back” functionality, affording a reset to factory settings or aprevious firmware or software version if the update does not proceedcorrectly.

Test Scan Procedure

In an exemplary embodiment of the present invention, an operationprocedure for the conducting of test scans may be summarised as follows(shown in FIG. 5): The user adds a fluid sample to the assay cartridge,and the assay cartridge is left for sufficient time for the assay todevelop [501]. Next, the user selects the “run test” option of thereader's user-interface [502]. The reader system lifts the cartridgeholster to a cartridge access position, opening the reader lid [201],and prompts the user to insert the assay cartridge [503]. Upon insertionof the cartridge, the holster is brought to a “home” position, and theuser is prompted to enter a patient identification (via the integratedtext entry device, or external barcode reader) [504]. The system carriedout a scan of the assay panel, and calculates assay results [505]. Thecartridge holster is brought back to the access position, opening thereader lid, and the user is prompted to remove the cartridge [506].Finally, results are displayed on the reader screen, and areautomatically saved [507].

Liquid Calibrator Scan Procedure

In an exemplary embodiment of the present invention, the operationprocedure for the conducting of liquid calibrator scans to account forassay changes over time may be summarised as follows (shown in FIG. 7):The user adds a “level one” liquid control sample (with a characterisedconcentration of each analyte) to a standard assay cartridge of thebatch to be corrected, and the assay cartridge is left for sufficienttime for the assay to develop. Next, the user selects the “run liquidcontrol” option of the reader's user-interface [701]. The reader systembrings the cartridge holster to an access position, opening the readerlid, and prompts the user to insert the assay cartridge [702]. Thesystem carried out a scan of the cartridge assay panel, and processesthe raw data [703]. The cartridge holster is brought back to the accessposition, opening the reader lid, and the user is prompted to remove thecartridge [704].

If the assay batch corresponds to a quantitative assay panel, the useris then prompted to add a “level two” liquid control sample (with asecond characterised concentration of each analyte) to a second standardassay cartridge of this batch. The user leaves the assay cartridge forsufficient time for the assay panel to develop, before inserting theassay cartridge into the reader system [705]. The system carried out ascan of the assay panel, and processes the raw data [706]. The cartridgeholster is brought back to the access position, opening the reader lid,and the user is prompted to remove the cartridge [707].

In the case of either a qualitative or quantitative assay batch, resultsare then calculated from the processed raw data, displayed on the readerscreen, and automatically saved [708].

Scan Processing

In embodiments of the present invention, raw scan response data acquiredduring either test scans or liquid control scans are processed prior toestimation of analyte presence or concentration. This processingcalibrates the data to account for reader response, which may besomewhat different between reader channels, or between reader models.Following this, peaks are detected in the reader calibrated data,according to peak detection parameters stored within corresponding batchcalibration files. Finally, the data is checked for read or assay runerrors.

In an exemplary embodiment of the present invention, the algorithmutilised for the processing of raw scan data, following a test scan orliquid calibrator scan, but prior to assay calibration or calculation ofliquid calibrator results may be summarised as follows (as shown in FIG.8): Initially the raw data, corresponding to optical energy collected bya reader photosensor at points across the scan length [801], is scaledby an internal reader calibration function [802]. This function isgenerally of the form of a linear equation, and normalises each point ofthe data set accounts for inter- or intra-reader variability, and isstored in non-volatile memory within the reader system itself.

Next, analysis of peaks within the reader calibrated scan data iscarried out. A scan of a single cartridge channel may incorporate acontrol capture zone peak and any number of mobilizable reagent capturepeaks. Generally, each mobilizable reagent capture peak is associatedwith a separate assayed analyte within the fluid sample. An examplecalibrated scan is shown in FIG. 16 for a single channel. This figureshows a peak corresponding to the control reagent capture zone [1601],and a peak corresponding to photoluminescence from the mobilizablereagent capture zone [1602]. Generally, the reader system incorporatesan algorithm for the detection of optical emission peaks, correspondingto labelled mobilizable or control reagent capture zones from eachoptical scan. Algorithm parameters may include such details as expectednumbers of peaks, expected scan positions of peaks, expected widths ofpeaks, and expected ranges of peak heights. Peak detection algorithmsmay include background subtraction; compensating for backgroundfluorescence derived from the assay materials, stray background light,unbound labelled assay materials or other sources [1603]. This may berealised by subtracting the minima of a scan, or estimation ofbackground luminescence at the scan position of the photoluminescencepeak maximum. In an embodiment, such estimation is carried out byregistering luminescence levels at particular scan positions at adefined distance to either side of the peak position, determining alinear fit to the background versus scan position, and then estimatingthe level of background fluorescence at a scan position corresponding tothe peak maximum.

In a particular embodiment of the current invention, an algorithm isincorporated which searches for specific peaks within the optical scandata. The block-diagram operation of this algorithm is shown in FIG. 8.Parameters for the peak recognition are provided in the lot calibrationfile, which is generally stored in reader memory. Initially, thealgorithm processes the reader calibrated data [802] by determining thedifferential of this data [803]. Next, the differential is smoothedusing a Savitsky Golay smoothing filter [804]. Following this, each peakis detected by searching for corresponding rising edges, falling edgesand zero-crossing points within the smoothed differential data; withsearch parameters in accordance with those stored within the batchcalibration file [805]. The algorithm analysis derives the positions ofthe scan maxima for each peak. These positions may be further refined byinterpolation. Subsequently, a maximum response value is searched for inthe original scan response data [806]. This search is carried outbetween scan positions corresponding to the smoothed differential'srising and falling edges, respectively.

A background luminescence baseline is estimated by fitting a linearfunction between two points [807]. These points correspond to averagevalues of luminescence about two positions at set distances either sideof the corresponding peak maximum. Finally, the background correctedpeak height is estimated by subtracting the value of this linearfunction at the position of the scan maximum from the peak maximumitself [808]. This algorithm is carried out for each peak in the scandata.

In particular embodiments of the present invention, prior to running ascan, the reader software initially verifies the date of testing asbeing prior to the expiry date of a particular assay, and that the testdate and time being within a set period of time since the reader opticalquality control checks, or liquid control verification or calibrationcorrections of the cartridge batch in question. In addition, sets ofquality controls may be actualised in software to verify that the assayran in a defined manner. These may include: quality control check ofscan data, including a check of control line development, a check ofchannel clearance, and checks as to the size and position of peaks. Inparticular, the level of detected luminescence is characterised at aparticular scan position, defined in calibration parameters for theassay in question, at which no capture or control zones are present, andwhich generally corresponds to background fluorescence. If thisluminescence is found to be above a certain level defined in calibrationparameters for the assay in question, the unbound luminescent materialsis not taken to have achieved full clearance, and the particular assayis termed a “Missrun” [809]. Control zone peaks are further analysed: ifthese are not found, or are of insufficient magnitude, the assay islikewise is considered to have not fully developed, and is likewisetermed a “Missrun” [810]. In either case, no estimate is made of analyteconcentration or presence. Following this, the algorithm verifies that acapture zone peak was detected. If this is not the case, the algorithminterprets the magnitude of the capture zone peak to be negligible and acapture zone peak height value of “0” is utilised for assay calculations[811]. Finally, the results are output for analysis by the relevant testscan calibration algorithm, or liquid control algorithm [812].

Algorithm for Calibration of Test Scans

In an embodiment of the present invention, the reader includes acalibration algorithm for the qualification or quantification ofluminescence from active areas of an assay scan. This algorithm takes asinput the following: calibration parameters specific to the assay batchand peak heights as determined by a peak detection algorithm for each ofthe capture and control zones. For each analyte, the algorithm processesthe corresponding capture zone peak height, according to the calibrationparameters. Generally for qualitative tests, the algorithm compares ascaled peak height versus a threshold value, and reports a “positive” or“negative” test result. Alternatively for quantitative tests, thealgorithm characterises the concentration of an analyte within the testsample, according to assay specific calibration parameters. In thiscase, the algorithm may report that the concentration is less than aparticular limits of detection, or greater or less than particularlimits of quantization, respectively. Finally for semi-quantitativetests, the algorithm characterises the concentration of an analytewithin the test sample to be within specific ranges, according to assayspecific calibration parameters. It should be understood that amultiplex assay panel may consist of a selection of qualitative,quantitative and semi-quantitative assays, all within a singlecartridge, being read and interpreted simultaneously.

In an exemplary embodiment of the present invention, the test scancalibration algorithm may be summarised by FIG. 9. Taking as inputs thepeak height and error check data [901], and assay calibration parametersas given in the corresponding cartridge batch calibration file, thealgorithm determines the nature of the assay—either as a quantitative orqualitative assay [902]. In the case of a qualitative assay, thealgorithm may use a linear equation for calibrating the capture zonepeak height to account for variations in the assay batch response overtime [903]. The coefficients of this calibration function are stored inthe corresponding cartridge batch calibration file. The scaled responseis subsequently compared with the response threshold value [905], whichis also stored in the corresponding cartridge batch calibration file. Inthe case of a competitive assay, if the response is below thisthreshold, the assay is reported as positive for the analyte inquestion. Otherwise, the assay is reported as negative. In the case of asandwich assay, if the response is below this threshold, the assay isreported as negative for the analyte in question. Otherwise, the assayis reported as positive.

In the case of a quantitative assay, the algorithm may use a 5-parameterlog-logistic equation to estimate the analyte concentration from thecapture zone peak height.

Initially, the capture zone peak height is analysed to ensure it iswithin the range of the calibration 5-parameter log-logistic curveequation [904]. If the capture zone peak height is within the range ofthe calibration equation, calibration is carried out with regard to the5-parameter log-logistic equation, and the estimated analyteconcentration determined [906]. Otherwise, the concentration may bereported as beyond the respective limit of quantization.

The estimated analyte concentration is next compared against the lowerconcentration limit of quantization, and the upper concentration limitof quantization, as given in the cartridge batch calibration file forthe assay in question [907]. If the estimated concentration is outsideone of these bounds, reporting is carried out as: If the calculatedconcentration is below a stipulated lower limit of quantization, theresult is given as below this limit, rather than the estimatedconcentration. Conversely, if the calculated concentration is above astipulated upper limit of quantization, the result is given as abovethis limit, rather than the estimated concentration.

Finally, the respective results, specifically run errors and presence orconcentration estimation results are reported to the user, and theseresults saved [908].

Liquid Control Algorithms

In an embodiment of the present invention, the reader includes aprocessing algorithm for the updating of assay specific calibrationparameters to compensate for assay-related changes in mobilizablereagent capture zone luminescence response. This algorithm analyses theresponse of one or more assay cartridges of the specified batch run withcontrol liquids of specified analyte concentrations. Internalcalibration parameters are then updated to provide a best-fit result tocontrol responses.

In an embodiment of the present invention, liquid calibrators are usedto account for minor assay and reader changes over time. These aregenerally run and analysed by the corresponding liquid control algorithmon a monthly basis for each batch of assays. However, the requiredfrequency of this correction may be set by the administrator level user.

In an exemplary embodiment of the present invention, one stable controlliquid is run to recalibrate a single assay batch for qualitativeassays. This control liquid contains defined concentrations of theanalytes of interest. Generally, these concentrations are selected tocorrespond to the concentration thresholds for each of the analytes. Aspecific quantity of each control liquid is run in an individual,standard cartridge of that batch. Conversely for quantitative assays,two stable control liquids are run to recalibrate a single assay batch.Each of the two liquid controls contains defined concentrations of theanalytes of interest. Generally, the two concentrations of each analyteare selected to correspond with defined low and high concentrations,respectively, with mobilizable reagent capture zone luminescenceresponses within the linear range of the 5-parameter log-logistic curveequation. A specific quantity of each control liquid is run in anindividual, standard cartridge of that batch.

An example of a liquid control calibration adjustment algorithm for aqualitative test is shown in FIG. 11. Upon acquisition and processing ofscan data from the “level one” liquid control [1101], this data isverified to ensure that no miss-run has occurred, and that the capturezone peak of each assay analyte is within an expected range [1102]. Ifeither of these checks is failed, the user is prompted to repeat theliquid control calibration adjustment.

If these checks are passed for all assays, a new linear equation forcalibrating the capture zone peak height to account for variations inthe assay batch response over time is calculated.

Finally, the calibration file is updated with the new value of thelinear equation, and the result of the liquid calibration displayed forthe user.

An example of a liquid control calibration adjustment algorithm for aquantitative test is shown in FIG. 12. Upon acquisition and processingof scan data from the “level one” (e.g., low concentration levels)liquid control [1201], this data is verified to ensure that no missrunhas occurred. Next, the estimated concentration of analyte iscalculated; in an identical manner to that of a standard test scan. Forthis purpose, the original values for 5-parameter log logistic equationare used, as given in the batch calibration file. This equation is priorto corrections carried out in previous liquid controls, and this stepverifies the assay is still operating in a similar manner to that of thefreshly manufactured batch. This estimated concentration is thenvalidated, verifying this is within a set range, of the actualconcentration of the “level one” liquid control (also given in the batchcalibration file). If no error has occurred, and the estimatedconcentration is within the expected range, the raw and processed peakdata is saved, and the “level two” liquid control calibrator is calledfor. Alternatively, the user is prompted to repeat the “level one”liquid control calibrator [1202].

Upon acquisition and processing of scan data from the “level two” (e.g.,high concentration levels) liquid control [1203], this data is verifiedto ensure that no missrun has occurred. Next, the estimatedconcentration of analyte is calculated; in an identical manner to thatof a standard test scan. For this purpose, the original values for5-parameter log logistic equation are used, as given in the batchcalibration file. This estimated concentration is then validated,verifying this is within a set range of the actual concentration of the“level two” liquid control (also given in the batch calibration file).If an error has occurred, or if the estimated concentration is outsidethe expected range, the user is prompted to repeat the “level two”liquid control calibrator [1204]. Alternatively, the algorithmcalculates updated calibration parameters as below [1205].

Generally, the calculation of liquid control updated 5-parameter loglogistic parameters is carried out by the minimisation of errorresiduals.

Upon calculation of optimised calibration parameters, the batchcalibration file is updated to include these parameters [1206], and thesuccess of the liquid controls calibration adjustment algorithm isreported to the user [1207].

Quality Control Check

In particular embodiments of the present invention, the reader systemincorporates an optical quality control algorithm. This algorithmcompares the response from a quality control component scan with theexpected response, and thereby validates the reader for the analysis ofassays. This optical quality control may be required to be run atspecific intervals, such as to ensure daily optical checking of thereader operation. Parameters for this optical self check are stored inthe system's internal memory, and contain quality control verificationparameters for the specific reader system, as determined during readerverification. This optical quality control data file comprises specificinformation, such as: quality control cartridge barcode identifier, scanpositions of quality control features, and expected response range ateach quality control feature. Data corresponding to this file may beencoded on a 2-D barcode on the quality control component, or encodedwithin an RFID chip associated with the quality control component. Thisdata may be thereby read by the reader system, and stored in internalmemory.

In an exemplary embodiment of the present invention, a separatebar-coded optical quality control component is used and the optical selfcheck procedure may be described as follows (as shown in FIG. 6): Uponinitiation of the optical self check by the system operator [601] andinsertion of the corresponding quality control device [602], the systemchecks the device barcode to ensure this corresponds with the qualitycontrol barcode stored in the quality control parameters file in memory.If this file do not exist, or if the cartridge is incorrect the check ishalted.

The system then initiates a scan, recording the opticalphotoluminescence from the quality control cartridge according to thestandard scan procedure [603]. The optical quality control algorithmexamines the detected photoluminescence response at each of the definedquality control features scan positions and compares these responses tothe expected response range at each quality control feature,respectively. If every response is within the expected response range,the quality control test is reported as a “pass”. If any level fallsoutside these thresholds, the test is a failure, and the scan and testsare repeated. Scans and analyses are repeated up to three times. If oneof these scans is a “pass”, this is reported. If all these fail, thequality control test is reported as a “fail”. The user is prompted toremove the QC cartridge [604]. Results are then displayed and scandetails for the final scan are stored within reader memory [605],including: header details (such as: time/date, cartridge identifier,user identifier, scan parameters and final number of scans taken), acopy of the parameter file, the detailed “pass”/“fail” status for eachquality control feature and original scan data.

The optical quality control algorithm is shown in FIG. 10, and may besummarised as follows: The algorithm takes as inputs the raw scan data[1001], and quality control parameters (as given in a quality controlfile, specific to the reader and quality control device). The algorithmrecords the detected photoluminescence response at a scan positionnominated within the quality control data file. This positioncorresponds to defined quality control feature on the quality controldevice. Next, the algorithm compares the response to the expectedresponse range for quality control feature [1002]. This range isspecific to the feature, quality control device, and reader device; andis specified in the quality control data file. If the response is withinthis range, the optical setup is considered to be well aligned to thisfeature, and the result is a “pass”. Otherwise, the quality control testis a “fail”. This process is repeated for all features on all readchannels of the quality control chip [1003]. The algorithm then outputsresults corresponding to the “pass”/“fail” properties of each test[1004].

LED Feedback Control

In an embodiment of the present invention, the excitation sourceintensity is controlled through the measurement scan. This is carriedout by monitoring the excitation source intensity using one or morededicated photosensors. Active feedback of this intensity signal ensuresthat the emission power of the excitation source remains constantthrough the scan. This is important as changes in excitation sourceemission power lead to direct changes in assay photoluminescence whichmay create inaccuracies in measurements of analyte presence.

In an embodiment of the present invention, the reader has excitationsources being a bank of six similar LEDs. In this case, excitation powervariation may be caused by, for example: power regulation variation, LEDdegradation over time, and thermal responses. Of these, a thermalresponse is particularly notable. As temperature increases or decreaseslinearly, the emission intensity of an LED decreases or increasesexponentially, respectively. In this case, two sets of three LEDs eachare optically isolated from each other using a baffle. A singlephotodiode is placed within each LED chamber, and monitors threecorresponding LEDs. During the scan of a cartridge, only one LED in eachisolated set of three LEDs is active at a specific time. Some portion ofthe emission from each LED is back-scattered or reflected from theaperture plate. This is monitored by the relevant photodiode. The LEDemission power is thus detected by observing the photodiode signal.Monitoring and optimisation of the LED emission is carried out prior tothe acquisition of assay luminescence, for each activation pulse of thecorresponding LED. For example, optimisation of LED power may proceedfor 25 ms, and then acquisition of assay luminescence may proceed for 15ms for each scan data point. Alternatively, LED stabilisation may becarried out for each LED prior to the commencement of a scan, an LEDapplied voltage is maintained at the relevant stabilized setpoint foreach LED throughout the scan.

Such optimisation of LED emission power is carried out as follows.

The applied voltage to each of the LEDs is individually controlled bythe reader software. The voltage applied to each LED is optimised by theLED feedback algorithm to stabilise the optical emission power at anexpected level. Initial levels for LED control voltages [1401] andexpected phototransistor response for the desired LED emission power arestored in the reader calibration file for each LED.

Initially, the LED is set at the initial default voltage [1401], and theLED monitoring optical signal read [1402]. Optimisations of LED controlvoltages are calculated using a control algorithm, such as aproportional-integral controller, separately for each LED [1404].

Print-Out of Data

In an embodiment of the present invention, the reader systemincorporates a printer [110] for the printout of hardcopies of scanresults and associated audit data following the reading of an assay, orfrom stored memory. A typical sample print-out following a scan of aqualitative, six analyte, drugs of abuse panel is shown in FIG. 13.Additional printable data may include: user lists, reader settings,events or error logs, installed calibrations, or quality controlresults.

In an embodiment of the present invention, touch-sensitive screenelements may be provided on the touchscreen interface [102] whichinitiate the printing of data, or the feeding of paper through theprinter. Further, prompts may be provided on screens in the userinterface containing printable material. These prompts inform the userthat these touch-sensitive screen elements may be used to initiateprinting of data.

System Self-Check

In embodiments of this invention, the reader incorporates software andelectronics for the initiation and interpretation of self-check tests.Generally, these tests may be initiated automatically at start up of thereader device, or initiated by selection of user interface options bythe user. Upon initiation of the self-check, the reader verifies theoperation of various internal and peripheral components. For example,verification may be carried out on memory devices, wirelesscommunications, port connections, motor operation, various controlsub-systems, excitation sources and detectors, printer operation,internal and external barcode sensors, battery operation, and powersupply operation.

Further connectivity may be provided for password protected access todevice test menus incorporating these and further test operations. Thesemay aid an engineer in the identification and resolution of errorsoccurring in device operation.

Upon encountering an error, this may be reported to the user, and adetailed report included in an internal events or error log file. Thereader may also be prevented from initiating tests while components havebeen found to be in an error state. Further reader functionality may belikewise restricted should associated components be detected to be in anerror state.

Security

In embodiments of the present invention, various strategies may beprovided to achieve security of data. For example, various user accesslevels may be provided; each with specific levels of data access andcontrol rights. One implementation of the current invention has twoaccess levels, being “administrator” and “user”. Generally, the “user”level has a subset of the “administrator” level rights. Specifically;scan, quality control, calibration, record review and printingfunctionality is available to all operators. In addition to theserights, the “administrator” level operator has access to additionalfunctionality, including the transfer of records to external devices,deletion of records, initiating of firmware or software updates, andsetting of reader options. In an embodiment of the present invention,the “administrator” level user can create and manage user accounts, setrequirements for entering passwords at log-on, and further set thesepasswords for each user.

In an embodiment of the present invention, event audit logs aremaintained of all settings changes, scans and system warnings anderrors. Each system event is uniquely identifiable, and is linked to thetime of the event, and the user logged into the device.

In an embodiment of the present invention, batch calibration files mayonly be acquired from specific secure, non-rewritable chips. Thesecalibration files may be encoded, to prevent interpretation outside thereader device.

In an embodiment of the present invention, connectivity to the reader,and access to reader connection menus may be further passwordcontrolled. Wireless connections from the reader device may requiresetup using the reader user interface by an “administrator” level user.

Reader Settings

In embodiments of the present invention, the reader user interfaceprovides settings sub-menus which enable an “administrator” level userto set or modify various reader settings and parameters. These mayinclude: configuration of user IDs and passwords; requirements for, orrequired frequency of, optical quality control or liquid control scansfor the running of test scans; language settings; display brightness;configuration of functions enabling the reader timing of assaydevelopment, and subsequent automatic initiation of scans; setting ofwireless connections and settings; handling of error reports; volumelevels of integrated speakers; management and/or deletion of savedresults and calibrations; or activation of ports and data communicationsettings.

Memory and Files

In embodiments of the present invention, the reader system incorporatesmemory and a file management system for the storage of essential data,operation parameters, and software and user interface details. Filesstored within this memory may include: Scan files, calibration files,quality control run files, user lists, settings and change logs, scanlogs, calibration run logs, or user logs. In order to review apotentially large volume of scan files, search functionality may beimplemented. This allows the user to filter scans results by date,operator, patient ID or test. Generally, original scan data andcalibration parameters are included in each scan results file, inaddition to salient reader information for quality control of scans.

In a particular embodiment of the present invention, the reader systemmay store five thousand patient scan records in internal memory, withthe oldest records being deleted once new ones are taken.

EXAMPLES

The following examples serve to further illustrate the methods anddevices of the present disclosure. These examples are in no way intendedto limit the scope of the invention.

Example 1: Six Channel Fluorescence Reader System

This example describes an example reader system for the recording andinterpretation of fluorescence from immunoassays, according to thepresent invention. This reader system receives a cartridge, comprisingsix vertical channels. Each channel comprises an immunoassay configuredfor the detection of a specific analyte within a single fluid sample.The reader system captures fluorescence from the surface of eachimmunoassay, quantifies the fluorescent response the single capture zoneof each immunoassay, and determines a quantitative or qualitativemeasurement each analyte's presence within said sample.

A system diagram of the reader is shown in FIG. 1. In addition, adiagram of the reader and the reader's optical components are shown inFIGS. 2 and 3, respectively. In brief, the reader system comprises acasing [204], incorporating a touch-screen display and data entry device[102] an on/off switch [101], a multicolor status indicator light [202]and a device access port with rotating lid [201]. The casing contains aholster receptacle [301] for receiving an external immunoassay device[107]. Further, the reader comprises an optical system within thecasing, consisting of excitation and collection optics within a singleoptical block; and an electromechanical motor system, such as a steppermotor, whereby the holster is moved with respect to the optical block[106].

Also, the reader system incorporates digital processors [104] andelectronics for the actuation and control of readings, and non-volatiledigital memory for the storing of data [105]. Finally, the readerincorporates communication ports and wireless connectivity [114], apower management system [113], an internal battery [111] and an externalthermal printer [124] for the printout of hardcopies of scan resultsfollowing the reading of an assay, or from stored memory. This printerinterfaces with and is powered by the reader via a communication port.

With regard to the particulars of the reader assembly and components,these are further detailed, below.

Within the cartridge holster [301], spring loaded dowels are located inpositions corresponding to recesses in the assay cartridge when thecartridge is correctly localised within the holster. Upon correctinsertion of the cartridge, the dowels register with the recessedfeatures of the cartridge. This locks the cartridge in position,ensuring assays are localised at the optical plane until a force isapplied to remove said cartridge. Also, physical alignment featuresprevent the mis-insertion of the cartridge, by blocking insertion of thecartridge at an incorrect rotational alignment.

The reader incorporates optical and mechanical sensors, which registerfull cartridge insertion and removal. These sensors are held within thecartridge holster, and their positioning corresponds to locations whichdefine the cartridge insertion or removal of the cartridge. In thiscase, the optical sensor is a light source and photosensor couple. Thisis be located in close proximity to the mouth of the holster. Uponinsertion, the cartridge blocks propagation of light from the sensorlight source to its corresponding photosensor. The sensor registers fullremoval of the cartridge by the resumption of light propagation from thesensor light source to its corresponding photosensor. A mechanicalswitch sensor is located at the base of the holster. Upon full insertionof the cartridge into the holster, this switch is actuated by thecartridge, enabling the detection of cartridge insertion.

The motor component integrates an encoder system which detects andreports the relative motor actuation position. Holster position may bedetermined with reference to this signal and signals received fromparticular optical travel sensors [120 a] located relative to specificpositions in the holster travel. The holster component incorporates beamblocking features, which break an optical beam sensed by the opticaltravel sensors, indicating holster position at these locations.

The reader system incorporates an internal 2D barcode camera system[120] for the reading of barcode information encoded on the cartridge.This camera system further incorporates a light source for illuminationof the barcode within the reader.

The excitation optics within the optical block include: six lightsources [302], an aperture plate [303], an interference filter [304],and a single excitation lens [305].

Light sources consist of surface mounted device LEDs, each with anintegrated lens which serves to partially collimate emitted light [302].The LEDs have an optical emission wavelength of circa 606 nm (LOE63B;Osram GmBH). The aperture plate is a thin flat metal shim, etched withsix rectangular optical apertures which restrict excitation light raysto those passing through the aperture [303]. Each aperture is aligned toemission from a single LED. In the reader system, apertures are 0.7mm-0.8 mm in width and 0.3-0.4 mm in height, and each excitation area is1.2 mm in width and 0.6 mm in height.

The excitation filter [304] is located within the light paths ofexcitation and centred within these light paths using a tubeconstruction system. The optical excitation filter shapes the spectralprofile of excitation light experienced by the immunoassay device. Thisfilter acts to ensure spectral separation between excitation light andphotoluminescent label emitted light. An optical excitation filter may,for example, be of band-pass or short-pass variety, and may operate byinterference or absorptive mechanisms. Generally, the excitation filteris selected such that the filter pass-band corresponds to some portionof the excitation spectrum of the photoluminescent label, and that thefilter stop-band corresponds to some portion of the emission spectrum ofthe photoluminescent label. The Stokes' shift between thephotoluminescent label's excitation and emission spectra defines themaximum filter transition band. In this case, a band-pass interferenceoptical filter is selected, with a central pass wavelength of 590 nm,and a transparent bandwidth of 60 nm (BK-590-60; Interferenzoptik GmbH).

The excitation lens [305] is located within the light paths ofexcitation and centred within these light paths using a tubeconstruction system. This lens directs light source emitted opticalenergy to the surface of the immunoassay device. The lens is biconvexaspheric in design, and is formed of transparent cyclic olefinco-polymer material (Zeonex 480 R).

Generally, the light sources have emission wavelengths compatible withthe excitation spectra of photoluminescent labels associated with themobilizable or control reagents of the assay. Such labels may includedark red emitting fluorophores, such as DyLight® 650 (Thermo-FischerScientific), Alexa Fluor® 647 (Invitrogen Corporation) or Cy5.

Collection optics within the optical block include: six collectionlenses [307], a glass absorptive filter [308] and six photodiodes forthe detection and quantification of this luminescence [306]. Eachcollection lens [307] collects light from an individual excitation area,an area of the immunoassay surface illuminated by the excitation opticalassembly; and directs this light towards the central portion of acorresponding photodiode [306]. All six lenses are identical in design,being biconvex aspheric, and are formed of transparent cyclic olefinco-polymer material (Zeonex 480 R). A single long-pass glass absorptiveoptical filter [308] is used to filter excitation light from allchannels. This filter has optical pass-band beyond a wavelength of ca.665 nm (ZVL050; Knight Optical (UK) Ltd.). This filters out residualreflected or scattered excitation light, and passes light associatedwith fluorescence of the labelled conjugates.

There is an angular offset between the plane of optical collection pathsof the collection optics, and the plane of optical excitation paths ofthe excitation optics; with the optical excitation plane is normal tothe cartridge surface, while detection is offset by 35 degrees. Theangular position of these planes and their specific offset are selectedin order to inhibit direct reflection of excitation light into thedetector assembly.

The reader system incorporates digital processors and electronics forthe actuation and control of readings. Generally an operations processor[104 a] controls time critical sensing and control operations, such asthe operation of motors, optical electronic components, sensors, andscan processing. An additional interface processor controls display andinterface components, interpreting data entry and communicationsprotocols. Additionally, this processor control internal digital memory[105]; enabling the writing, reading, search and transfer of data.

The reader system incorporates non-volatile digital memory for thestoring of data [105]. Generally, such data includes collected scandata, and corresponding patient details and assay results; user detailsand passwords; events and error logs; calibration parameters; readersettings; user interface screens; interface and communicationsparameters; and reader operation programs. This memory consists of:internal flash memory and an internal SD-card.

The reader system incorporates communications ports [114]. Inparticular, components and protocols are incorporated for wiredconnectivity, including USB, and Ethernet. These facilitatecommunication to, and control of, devices external to the reader.Specifically, this connectivity enables remote diagnostics, firmware orsoftware updates and data transfer, and control of an external barcodereader device [115]. The reader also includes components and protocolsfor external wireless access by WI-FI. Specifically, this connectivityenables remote diagnostics, firmware or software updates and datatransfer.

The reader system incorporates an external thermal printer [124] for theprintout of hardcopies of scan results and associated audit datafollowing the reading of an assay, or from stored memory. This printeris communicates and is powered by the reader via a connection to one ofthe readers communication ports [114]. The reader system is portable,being intended for bench- or table-top point-of-care use, and includesan internal battery [111], which can power the reader in situationswhere the system is not connected to a power supply [112]. This batteryis rechargeable, and recharges while the reader is connected to a mainspower supply. A power system [113] monitors battery charge, reportingthis to the user, and regulating such details as: charge speed, batterytemperature, and minimum charge levels before the unit is automaticallyshut down.

In some embodiments, an SD card component holds assay specificcalibration data relating to an assay cartridge batch. The SD card maybe introduced into a reader system socket, and the assay specificcalibration data copied to internal reader memory. SD card devices areof a secure write-once, read many times form. Additionally, a standardSD may be inserted into the SD slot, and the user may transfer saveddata (such as scans, results, settings, calibrations or quality controldata) from the internal device to the SD card for back up or subsequenttransport.

With regard to system control, the LED emission timings and photodioderead timings are tuned such that only one test is being excited at aspecific time, ensuring that optical crosstalk between channels isminimised. Further, the LED emission intensity for each LED isstabilized to a standard setpoint prior to the commencement of each scanthrough the measurement scan. This is carried out by monitoring theexcitation source intensity using two dedicated photodiodes. Activefeedback of this intensity signal ensures that the emission of theexcitation source remains constant across all scans.

Also, prior to each LED emission pulse, the reader system also records adark count, corresponding to detected signal without activation of thecorresponding LED. This is integrated over an equivalent time to the LEDpulse time. Following the recording of all scan points, the receivedsignals are corrected by subtraction of each dark count from thatacquired during activation of the corresponding LEDs. This enablescompensation for light leakage into the device, interference from lightgeneration within the device, or thermal or electronic noise. The readerincludes an algorithm for the detection of optical emission peaks fromeach optical scan. Algorithm parameters may include such details asexpected numbers of peaks, expected peak scan positions, expected widthsof peaks, expected ranges of peak heights. The peak detection algorithmincludes background subtraction; compensating for backgroundfluorescence derived from the assay materials, stray background light,unbound labelled assay materials or other sources. This is realised byestimation and subtraction of background fluorescence at the point ofthe peak maximum. Such estimation is carried out by registeringfluorescence levels at particular scan positions at a defined distanceto either side of the peak position, then determining a linear fit tothe background versus scan position, and then estimation of the level ofbackground fluorescence at a scan position at a position correspondingto the peak maximum.

Sets of quality controls are actualised in software to verify that anassay panel ran in a defined manner. These include: quality controlcheck of scan data, including a check of control line development, acheck of channel clearance, and checks as to the size and position ofpeaks. Additionally, the software verifies the time of test as beingwithin the expiry data of a particular assay. In particular, the levelof detected luminescence is characterised at a particular scan position,defined in calibration parameters for the assay in question, at which nocapture or control zones are present, and which generally corresponds tobackground fluorescence. If this luminescence is found to be above acertain level defined in calibration parameters for the assay inquestion, the unbound luminescent materials is not taken to haveachieved full clearance, and the particular assay is termed a “Missrun”.Control zone peaks are further analysed: if these are not found, or areof insufficient magnitude, the assay is likewise is considered to havenot fully developed, and is likewise termed a “Missrun”.

The reader includes a calibration algorithm for the qualification orquantification of luminescence from active areas of an assay scan. Thisalgorithm takes as input the following: calibration parameters specificto the assay batch and peak heights as determined by a peak detectionalgorithm for each of the capture and control zones. For each analyte,the algorithm processes the corresponding capture zone peak height,according to the calibration parameters. Generally for qualitativetests, the algorithm compares the peak height versus a threshold value,and reports a positive or negative result. Alternatively forquantitative tests, the algorithm characterises the concentration of ananalyte within the test sample, according to assay specific calibrationparameters. Should the estimated concentration be outside the assay'sbounds of quantization, the algorithm reports that the concentration isgreater than or less than particular limits of quantization,respectively.

A physically separate quality control component [108], of externaldimensions similar to that of the assay cartridge is incorporated. Thiscomponent incorporates materials exhibiting specific, characterisedlevels of fluorescence. The quality control component's fluorescentareas are defined using masked fluorescent PVC materials. The qualitycontrol component's fluorescent areas may be localised at the opticalplane within the reader. Fluorescent areas are patterned in a definedmanner, such that optical misalignments lead to predictable changes inscan responses. A processing algorithm for the analysis of qualitycontrol component scans is incorporated in the reader. This algorithmcompares expected responses from fluorescent areas with those ofreceived responses, and validates the reader for the analysis of assays.

The reader also includes a processing algorithm for the verification ofassay batch responses or optionally, the updating of assay specificcalibration parameters to compensate for assay based changes inresponse. This algorithm analyses the response of one or more assaycartridges of the specified batch run with control liquids of specifiedconcentrations. Assay responses are verified to be within expectedlimits. Additionally, internal calibration parameters may then beupdated to provide a best-fit result to control responses.

1-55. (canceled)
 56. A reader system for analyzing one or more analytesin a fluid sample, the reader system comprising: a casing; a verticallyoriented holster within the casing, wherein the casing comprises atleast one port leading to the vertically oriented holster; an insertableand removable cartridge placed in the vertically oriented holster,wherein the cartridge comprises a vertically oriented immunoassay devicefor analyzing one or more analytes in a fluid sample; an optical system,comprising: excitation optics comprising at least one light source andan excitation lens, wherein the excitation optics transmit excitationlight from the at least one light source in a direction that ishorizontal relative to the vertically oriented holster and verticallyoriented immunoassay device, and wherein the transmitted light excites aregion of the vertically oriented immunoassay device, and collectionoptics comprising at least one photosensor and at least one collectionlens, wherein the collection optics collect light from the excitedregion of the vertically oriented immunoassay device; anelectromechanical motor system, wherein the electromechanical motorsystem moves the vertically oriented holster; and one or more digitalprocessors, and associated electronics configured to receive data fromand control the optical system and to control the electromechanicalmotor system.
 57. The reader of claim 56, further comprisingnon-volatile or volatile digital memory for storing data generated bythe optical system.
 58. The reader system of claim 56, wherein thecasing further comprises a display screen, a data entry device, or acombination thereof.
 59. The reader system of claim 58, wherein the dataentry device comprises an integrated keypad or a touch-screen.
 60. Thereader system of claim 56, wherein the at least one light source is asurface mounted light emitting diode.
 61. The reader system of claim 60,wherein the surface mounted light emitting diode comprises a collimationlens.
 62. The reader system of claim 56, wherein the at least onecollection lens is configured to collect emitted light from an entireexcited region.
 63. The reader system of claim 62, wherein emitted lightis directed onto the central portion of a photodiode.
 64. The readersystem of claim 56, wherein the excitation optics further comprises anoptical excitation filter.
 65. The reader system of claim 56, whereinthe collection optics further comprises an optical collection filter.66. The reader system of claim 65, wherein the optical collection filteris mechanically actuated.
 67. The reader system of claim 56, wherein theexcitation optics further comprises a plate with one or more opticalapertures.
 68. The reader system of claim 56, wherein the verticallyoriented immunoassay device comprises one or more immunoassay channels,each channel comprising one or more test lines for analyzing one or moreanalytes in the fluid sample.
 69. The reader system of claim 56, whereinthe vertically oriented immunoassay device comprises multipleimmunoassay channels and the excitation optics comprises multiple lightsources, each with different central wavelengths.
 70. The reader systemof claim 69, wherein the collection optics comprises multiplephotosensors, and the multiple light sources and photosensors areconfigured in pairs so that they transmit light to and collect emittedlight from different immunoassay channels.
 71. The reader system ofclaim 69, wherein the pairs of light sources and photosensors areconfigured so that different immunoassay channels are interrogated atdifferent points in time.
 72. The reader system of claim 69, whereinelectronic frequency filtering is used to filter signals from themultiple photosensors.
 73. The reader system of claim 69, wherein themultiple immunoassay channels are spatially separated such that crosstalk between different immunoassay channels and different photosensorsis substantially absent when the multiple immunoassay channels areinterrogated simultaneously.
 74. The reader system of claim 56, whereinthe at least one photosensor monitors the at least one light source. 75.The reader system of claim 74, wherein measurements from the at leastone photosensor that monitors the at least one light source are used tocontrol an emission intensity of the at least one light source.
 76. Thereader system of claim 56, wherein an optical collection plane of thecollection optics and an optical excitation plane of the excitationoptics form an angle that is offset from normal.
 77. The reader systemof claim 56, further comprising at least one sensor for recognizingcartridge insertion or removal.
 78. The reader system of claim 77,wherein the at least one sensor comprises an optical or mechanicalsensor.
 79. The reader system of claim 56, further comprising a barcodereading system.
 80. The reader system of claim 79, wherein the barcodereading system is two-dimensional, and the two-dimensional barcodereading system is characterized in that it encodes information selectedfrom the group consisting of identification of cartridge type or lotdata, lot manufacture and expiry dates, analyte names, cartridgeexpected response, lot parameters, peak finding parameters, calibrationparameters, and any combination thereof.
 81. The reader system of claim56, wherein the electromechanical motor system comprises an encoderwhich is used to detect and report a relative position of the holster.82. The reader system of claim 56, further comprising alignment featureswithin the vertically oriented holster for ensuring alignment of thecartridge.